Diabetes Mellitus is characterized by a deficiency in insulin secretion and insulin resistance, or A deficiency in pancreatic β-cells resulting from autoimmune destruction. During the past 10 years, the diabetic population in the United States has increased 86%. It is estimated that over 23.6 million children and adults, or 7.8% of the population, had diabetes in 2008 (ADA, 2008), 90% of whom had type 2 diabetes. It is estimated that by the year 2030, the total number of diabetic people will rise to 366 million world-wide (Wild et al., (2004) Diabetes Care 27: 1047-1053). Although a number of treatments have been developed and have proved to be very effective (Riddle, M C (2002) Diabetes Metab. Res. Rev. 18 (Suppl. 3): S42-49), development of new treatment agents and strategies that provide lasting effects, have less drug side effects such as hypoglycemia and weight gain, and better drug administration routes, remain a major challenge in management of the disease.
Diabetes development and progression is often characterized by loss of pancreatic β-cells and β-cell functions (Stoffers, D A, (2004) Horm. Metab. Res. 36: 811-821; de Koning et al., (2008) Trends Pharmacol. Sci. 29: 218-227). The ability to assess the pancreatic islet and β-cell mass and functions would be greatly beneficial for diagnosis/prognosis of diabetes and understanding the pathogenesis of the diseases. Non-invasive assessment of pancreatic β-cells and their function will also enable better design of the disease treatment strategy and monitoring the effectiveness of therapies.
Very important progress has been made in imaging of pancreas (Holmberg & Ahlgren, (2008) Diabetologia 51: 2148-2154). However, there are several important challenges in the imaging of pancreatic islets, especially the islet β-cells. The pancreatic islets are small and distributed throughout the entire pancreas, this demanding a high resolution imaging method to clearly locate and estimate the β-cell mass in islets of the pancreas. The pancreas islet is a tissue mass of many endocrine cell types, including a-cell, β-cells, δ-cells, ε-cells, and PP cells, and these different types of cells are completely intermingled throughout the islet.
Imaging tools or agents targeting β-cell specific molecular markers are required to image β-cells in the pancreatic islets. No successful imaging method that allows non-invasive imaging of pancreatic β-cells is currently available.
Glucagon-like Peptide-1 (GLP-1), a 30 amino-acid peptide, is one of the major incretin hormonal intestinal-derived factors secreted to lower the blood glucose (Drucker, D J, (2006) Cell Metab. 3: 153-165). The peptide is produced in enteroendocrine L cells in small bowel and colon. GLP-1 is secreted by the L cells as a 37 amino acids precursor and the peptide is processed to a bioactive form of a 30 amino acid (7-37) amide (Baggio & Drucker (2007) Gastroenterology 132: 2131-2157; Burcelin et al., (2007) J. Nutr. 137(11 Suppl): 2534S-2538S). The circulation time of the activated GLP-1 is less than 2 minutes due to degradation by a ubiquitous protease enzyme dipeptidyl peptidase 4 (DPP-4) to the inactive form of amino acids 9-37 (Baggio & Drucker (2007) Gastroenterology 132: 2131-2157; Holst et al., (2008) Trends Mol. Med. 14: 161-168). Evidence suggested that the inactive (9-37) GLP-1 plays a role in clearance of glucose and regulation of cardiovascular function (Drucker, D J, (2006) Cell Metab. 3: 153-165; Mannucci & Rotella (2008) Nutr. Metab. Cardiovasc. Dis. 18: 639-645; Nauck, M A, (2009) Eur. J. Intern. Med. 20 (Suppl 2): S303-308). GLP-1 acts via a cell surface receptor, GLP-1 receptor (GLP-1R) belonging to the class B family of 7-transmembrane spanning heterotrimeric G-protein coupled receptors (Mayo et al., (2003) Pharmacol Rev. 55: 167-194).
GLP-1R is expressed in the pancreatic islet in very high levels (Korner et al., (2007) J. Nucl. Med. 48: 736-743). The receptor is also expressed in several other organ sites, including, kidney, heart, lung, and central nervous system (Doyle & Egan (2007) Pharmacol. Ther. 113: 546-593). In the pancreatic islet, GLP-1R is predominately located in the β-cells at a density as high as 105-106 receptormolecules/cell (Korner et al., (2007) J. Nucl. Med. 48: 736-743; Wei & Mojsov (1995) FEBS Letts. 358: 219-224).
The insulinotropic actions of GLP-1 include insulin secretion and insulin biosynthesis including proinsulin gene expression (Egan et al., (2003) Diabetes Metab. Res. Rev. 19: 115-123; Winzell & Ahren (2007) Pharmacol. Ther. 116: 437-448). This stimulation of insulin secretion in pancreatic β-cells by GLP-1 is dependent on elevation of plasma glucose. The detailed mechanism by which the GLP-1 stimulates insulin secretion under elevated plasma glucose is not well understood. GLP-1 may act via the GLP-1R to stimulate cyclic AMP formation and activate protein kinase A in pancreatic β-cells. The action of GLP-1 also includes replenishment of the intracellular insulin pool by up-regulating the expression of proinsulin, which includes proinsulin gene transcription and mRNA stability (Doyle & Egan (2007) Pharmacol. Ther. 113: 546-593; de Heer et al., (2008) Diabetologia 51: 2263-2270; Ahren et al., (2004) Horm. Metab. Res. 36: 733-734). It was demonstrated that the production of cAMP and activation of PKA under GLP-1 stimulation activates the transcription activator Pdx that plays an important role in insulin gene transcription (Li et al., (2005) Diabetes 54: 482-491). In addition to insulintropic actions, GLP-1 promotes differentiation of progenitor cells to mature β-cells in islet (Yue et al., (2006) Tissue Eng. 12: 2105-2116) and also trigger cellular processes in pancreatic β-cells that promote β-cell proliferation and inhibit apoptosis, which consequently leads to an increase in pancreatic β-cell mass and normalizes the β-cell function in pancreas (Doyle & Egan (2007) Pharmacol. Ther. 113: 546-593; Klinger et al., (2008) Diabetes 57: 584-593; Bonora, E, (2008) Nutr. Metab. Cardiovasc. Dis. 18: 74-83).
Rapid degradation of native GLP-1 by DPP-4 hampers the application of the native GLP-1 as a potential diabetes treatment. Substantial efforts were made to develop a diabetes treatment based on the GLP-1 and GLP-1R pathway (Ahren & Schmitz (2004) Horm. Metab. Res. 36: 867-876; Salehi & D'Alessio (2006) Cleve. Clin. J. Med. 73: 382-389; Arulmozhi & Portha (2006) Eur. J. Pharm. Sci. 28: 96-108; McGill, J B (2009) Postgrad. Med. 121: 46-45). Most attention has focused on developing GLP-1R agonists and DPP-4 inhibitors (Combettes, M M (2006) Curr. Opin. Pharmacol. 6: 598-605; Gromada et al., (2004) Basic Clin. Pharmacol. Toxicol. 95: 252-262).
One early GLP-1R agonist is exendin-4, a 39 amino acid GLP-1 analog, purified from the saliva of the lizard Heloderma suspectum. Exendin-4 is resistant to DPP-4 cleavage (Deacon, et al., (1998) Diabetologia 41: 271-278) resulting in an approximately 3 hour blood circulation time in human. Exendin-4 has been approved by the FDA as a treatment of type 2 diabetes by twice daily injections. However, short blood circulation time limits the effectiveness of the exendin-4 as diabetes treatment.
Several new agents acting as long-lasting GLP-1R agonists are currently undergoing clinical trials. Substitution of two amino acids of GLP-1 and acylation of the peptide with a long chain fatty acid led to development of Liraglutide (Knudsen, L B (2004) J. Med. Chem. 47: 4128-4134; Juhl et al., (2002) Diabetes 51: 424-429). Thus, mutations at two amino acids resulted in DPP-4 resistance and acylation led to the binding of the peptide to serum albumin. The resultant peptide has a more than 10 hr blood circulation time. Another DPP-4 resistant and long circulating GLP-1R agonist was developed by substituting the Ala-18 of GLP-1 with D-Ala and then linking it (via maleimidoproprionic acid) to the C-terminal of serum albumin (CJC-1131) (Kim et al., (2003) Diabetes 52: 751-759). Albugon is another serum albumin based GLP-1 agonist (Baggio et al., (2004) Diabetes 53: 2492-2500). In this case the GLP-1 is directly conjugated to the serum albumin.
Most approaches for development of long circulating GLP-1R agonists were based on binding or conjugation to serum albumin. These approaches achieved great successes, but, there are drawbacks. Serum albumin is a protein of about 70 kDa molecular mass. The large molecular size limits its capability of endothelial penetration and tissue penetration. Biodistribution of serum albumin also do not favor pancreas delivery of the agents. Intensive studies revealed that serum albumin stays in circulation for a long time with little up-taken by pancreas, especially by islets (Bent-Hansen, L, (1991) Acta. Physiol. Scand. Suppl. 603: 5-10). This property significantly limited the delivery of the agent in response to a transient elevation of blood glucose, such as the situation after meals. There is, therefore, an urgent need to develop GLP-1R agonists by alternative approaches.